Not applicable.
Not Applicable.
(1) Field of the Invention
The present invention relates to anthraquinones which are antihelminthic and in particular, are useful in compositions for inhibiting Schistosoma sp. in vitro or in vivo. The preferred anthraquinones have the formula: 
wherein R1, R2, R3, and R4 are each hydrogen, hydroxy, halogen, alkyl, substituted alkyl, alkene, substituted alkene, alkyne, aryl, substituted aryl, cyclic, substituted cyclic, acid group, carbohydrate, or combination thereof, R is a group containing 1 to 12 carbons such as methyl, alkyl, substituted alkyl, aldehyde, hydroxymethyl, acid and carbohydrate groups, and a hydroxy group or combination thereof, and the halogen is I, F, Br, or Cl. In a particular embodiment, the anthraquinones have the formula 
wherein R is a group containing 1 to 12 carbons such as methyl, alkyl, substituted alkyl, aldehyde, hydroxymethyl, acid carbohydrate, groups and a hydroxy group, and combinations thereof.
(2) Description of Related Art
Schistosomasis is a disease caused by parasitic digenetic trematodes of the genus Schistosoma that afflicts at least 200 million people worldwide with another 600 million at risk (Chitsula et al., Acta Trop. 77: 41-51 (2000)). Chronic Schistosoma infection can lead to the development of a variety of conditions including diarrhea, hepatic fibrosis and portal hypertension, central nervous system disease, embolisms of the pulmonary arterioles, and hematuria. While a large number of schistosomes are known, only five appear to be primarily responsible for human infections including Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma intercalatum, and Schistosoma haematobium. 
These digenetic schistosomes have a complex life-cycle in which free-swimming cercariae emerge from intermediate freshwater snail hosts and infect humans by attaching to the skin via an oral sucker or mucus secretion and penetrate the dermis by releasing proteolytic enzymes. Concurrently, the cercariae shed their tails and transform into schistosomula that enter the venous vascular system where they are carried to the heart and lungs before reaching the systemic circulation. Ultimately, the schistosomula arrive at the liver where they grow into sexually mature adults. Male and female adults form copulating pairs that migrate down the portal vein, eventually reaching the mesenteric or vesical veins, depending on the specific species of schistosome, and begin laying eggs for a period of typically 3 to 5 years. The eggs are generally responsible for triggering the host""s immune response that results in the formation of granulomas that lead to the sequelae of clinical manifestations (Bica et al., Infect. Dis. Clin. N. Am. 14: 637-642 (2000); Elliot, Gastroenterol. Clin. N. Am. 25: 599-624 (1996); Morris and Knauer, Sem. Respir. Infect. 12: 159-170 (1997); Schafer and Hale, Curr. Gastroenterol. Reports 3: 293-303 (2001)).
There are limited options available for the chemotherapeutic treatment for Schistosoma infections with the drug-of-choice being the pyrazionoisoquinoline, praziquantel (Elliot, ibid.). Unfortunately, the long-term worldwide application of the drug coupled with the recent discovery of praziquantel-tolerant schistosomes has generated concern over the development of drug-resistant Schistosoma strains (Cioli, Parasitol. Today 14: 418-422 (1998) and Curr. Opin. Infect Dis. 13: 659-663 (2000); William et al., Parasitol. 122: 63-66 (2001)). With few other options available for combating schistosomiasis, there is an urgent need to develop new methodologies for the treatment and prevention of Schistosoma infection (Cioli, ibid.).
Daylily roots (Hemerocallis spp., Hemerocallidaceae) have been used in Asia to treat schistosomiasis (Shiao et al., Acta Pharma. Sinica 9:218-224 (1962); Shiao et al., Acta Pharma. Sinica 9: 217-224 (1962)). However, this method of treatment has been disfavored due to a host of toxic side effects and deaths associated with the administration of Hemerocallis root extracts to humans (Wang et al., Phytochem. 28: 1825-1826 (1989)). Previous efforts to identify the active constituent responsible for the therapeutic properties of Hemerocallis roots lead to isolation of a neurotoxic binaphthalenetetrol known as stypandrol (Wang and Yang, 1993) which had been shown to cause paralysis, blindness and death in mammals (Main et al., Aust. Vet. J. 57: 132-135 (1981); Colegate et al., Aust. J. Chem. 38: 1233-1241 (1985)). In another report by Chen et al. (Acta Pharma. Sinica 9: 579-586 (1962)), researchers obtained a yellow powdery isolate to which the authors ascribed both the biological activity against schistosomes, as well as, the toxic side effects associated with the use of Hemerocallis roots; however, its structure was never identified. While other studies have described additional compounds found in daylilies, none of these efforts have addressed the need to fully characterize the bioactive schistosomicidal chemical constituents from Hemerocallis roots.
Compounds which have antihelminthic activity are known in the prior art such as oxamniquine, metrifonate, and 4-(4-nitroanilino)-phenylisothiocyanate, which are disclosed in U.S. Pat. No. 4,117,156 to Loewe et al. However, oxamniquine is only effective against Schistosoma mansoni, is more active against male rather than female worms, and has little effect on immature worms, and metrifonate is only active against Schistosoma haematobium. U.S. Pat. Nos. 5,091,385, 5,177,073, and 5,489,590 to Gulliya et al. disclose a therapeutic mixture comprising a photoactive compound which is capable of killing tumors, bacteria, viruses, and parasites such as Schistosoma when activated prior to use with an activating agent such as a chemical, radiation (preferably, irradiation with a laser), gamma rays, or electrons from an electropotential device. The photoactive compounds include a general suggestion of anthraquinones.
In light of the above, there remains a need for novel compounds with antihelminthic activity to increase the arsenal of drugs for combating helminthic infections in warm-blooded animals, including humans.
The present invention provides a method for inhibiting helminths such as those comprising the Schistosoma genus in vivo or in vitro by exposing the helminths to an inhibitory amount of one or more anthraquinones. The anthraquinones can be substituted with halogens such as I, F, Br, and Cl in the ring, particularly where hydroxyl groups are not located. The substituents in the ring can also include one or more of the halogens.
As used herein, the term xe2x80x9cinhibitoryxe2x80x9d means either to limit the growth of the helminth or cells, to stop the growth of the helminth or cells, or to kill the helminth or cells. Thus, the term embraces any affect which adversely affects the helminth or cells.
Therefore, in one embodiment, the present invention provides a method for inhibiting a parasitic helminth which comprises exposing the helminth to an inhibitory amount of an anthraquinone.
In particular, the present invention provides a method for inhibiting a parasitic helminth, which comprises exposing the helminth to an antihelminthic amount of at least one anthraquinone which has the formula: 
wherein R1, R2, R3, and R4 are each selected from the group consisting of hydrogen, hydroxy, halogen, alkyl, substituted alkyl, alkene, substituted alkene, alkyne, aryl, substituted aryl, cyclic, substituted cyclic, acid group, carbohydrate, and combinations thereof, R is a group containing 1 to 12 carbons selected from the group consisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acid and carbohydrate groups, and a hydroxy group and combinations thereof, and the halogen is selected from the group consisting of I, F, Br, and Cl.
The present invention further provides a method for inhibiting a parasitic helminth, which comprises exposing the helminth to an antihelminthic amount of at least one anthraquinone which has the formula: 
wherein R is a group containing 1 to 12 carbons selected from the group consisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxymethyl, acid and carbohydrate groups, and a hydroxy group, and combinations thereof.
The present invention further provides a method for inhibiting a Schistosoma sp. which comprises exposing the Schistosoma sp. to an inhibitory amount of at least one anthraquinone of the formula: 
wherein R1, R2, R3, and R4 are each selected from the group consisting of hydrogen, hydroxy, halogen, alkyl, substituted alkyl, alkene, Substituted alkene, alkyne, aryl, substituted aryl, cyclic, substituted cyclic, acid group, carbohydrate, and combinations thereof, R is a group containing 1 to 12 carbons selected from the group consisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxymethyl, acid and carbohydrate group, and a hydroxy group, and combinations thereof, and the halogen is selected from the group consisting of I, F, Br, and Cl.
The present invention further provides a method for inhibiting a Schistosoma sp. which comprises exposing the Schistosoma sp. to an inhibitory amount of at least one anthraquinone of the formula: 
wherein R is a group containing 1 to 12 carbons selected from the group consisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acid and carbohydrate groups, and a hydroxy group and combinations thereof.
In a preferred embodiment of the above methods, the anthraquinone is 1,2,8-trihydroxy-3-methyl anthraquinone (compound 3), 1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6), or both and the inhibiting can be either in vivo or in vitro.
In a further embodiment of the above methods, the anthraquinone is inhibitory at a dosage of 1 to 1,000 micrograms per milliliter or gram.
The present invention further provides a method for inhibiting a pathogenic trematode in a warm-blooded animal or human infected with the pathogenic trematode comprising (a) providing a composition containing an inhibitory amount of at least one anthraquinone selected from the group consisting of 1,2,8-trihydroxy-3-methyl anthraquinone (compound 3) and 1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6) in a pharmaceutically acceptable carrier; and (b) and administering the composition to the warm-blooded animal or human to inhibit the pathogenic trematode. A particular composition is a topical composition for swimmers itch which is a species of Schistosoma.
In a further embodiment of the method, the anthraquinone is inhibitory at a dosage of 1 to 1,000 micrograms per milliliter or gram.
In a further still embodiment of the method, the anthraquinone is administered to the warm-blooded animal or human orally, subcutaneously, intraperitoneally, topically, intravenously, topically, intranasally, or rectally.
The present invention further provides a method for inhibiting a pathogenic trematode in a warm-blooded animal or human infected with the pathogenic trematode comprising (a) providing a composition containing an inhibitory amount of 1,2,8-trihydroxy-3-methyl-O-xcex2-D-glucopyranoside anthraquinone (compound 7) and at least one anthraquinone selected from the group consisting of 1,8-dihydroxy-2-O-xcex2-D-glucopyranoside anthraquinone (compound 4) and 1,8-dihydroxy-2-O-malonyl-(1xe2x86x926)-xcex2-D-glucopyranoside anthraquinone (compound 5) in a pharmaceutically acceptable carrier; and (b) and administering the composition to the warm-blooded animal or human to inhibit the pathogenic trematode.
In a further embodiment of the method, the composition further includes an inhibitory amount of at least one anthraquinone selected from the group consisting of 1,2,8-trihydroxy-3-methyl anthraquinone (compound 3) and 1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6).
In a further still embodiment of the method, the anthraquinone is inhibitory at a dosage of 1 to 1,000 micrograms per milliliter or gram.
In a still further embodiment, the anthraquinone is administered to the warm-blooded animal or human orally, subcutaneously, intraperitoneally, topically, intranasally, intravenously, or rectally.
In a preferred embodiment of the above methods, the anthraquinone is selected from the group consisting of 1-hydroxy-2-acetyl-3,6-methyl anthraquinone (compound 1), 2-acetyl-3,6-methyl anthraquinone monoacetate (compound 1a), 1-hydroxy-2-acetyl-3,7-methyl anthraquinone (compound 2), 2-acetyl-3,7-methyl anthraquinone monoacetate (compound 2a), 1,2,8-trihydroxy-3-methyl anthraquinone (compound 3), 1,8-dihydroxy-2-O-xcex2-D-glucopyranoside anthraquinone (compound 4), 1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6), and 1,8-dihydroxy-3-carboxy anthraquinone (compound 8) and the inhibiting can be either in vivo or in vitro.
In a further embodiment of the above methods, the anthraquinone is inhibitory at a dosage of 1 to 1,000 micrograms per milliliter or gram.
The present invention further provides an antihelminthic composition which comprises (a) at least one anthraquinone which has the formula: 
wherein R1, R2, R3, and R4 are each selected from the group consisting of hydrogen, hydroxy, halogen, alkyl, substituted alkyl, alkene, substituted alkene, alkyne, aryl, substituted aryl, cyclic, substituted cyclic, acid group, carbohydrate, and combinations thereof, R is a group containing 1 to 12 carbons selected from the group consisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxy, hydroxymethyl, acid and carbohydrate groups, and a hydroxy group and combinations thereof, and the halogen is selected from the group consisting of I, F, Br, and Cl; and (b) a pharmaceutically acceptable carrier, preferably wherein the composition contains between about 1 and 1,000 micrograms of the anthraquinone per milliliter or gram of the carrier.
Preferably, the anthraquinone has the formula: 
wherein R is a group containing 1 to 12 carbons selected from the group consisting of methyl, alkyl, substituted alkyl, aldehyde, hydroxymethyl acid carbohydrate groups, and a hydroxy group, and combinations thereof.
More preferably, the anthraquinone is selected from the group consisting of 1-hydroxy-2-acetyl-3,6-methyl anthraquinone (compound 1), 2-acetyl-3,6-methyl anthraquinone monoacetate (compound 1a), 1-hydroxy-2-acetyl-3,7-methyl anthraquinone (compound 2), 2-acetyl-3,7-methyl anthraquinone monoacetate (compound 2a), 1,2,8-trihydroxy-3-methyl anthraquinone (compound 3), 1,8-dihydroxy-2-O-xcex2-D-glucopyranoside anthraquinone (compound 4), 1,2,8-trihydroxy-3-hydroxymethyl anthraquinone (compound 6), and 1,8-dihydroxy-3-carboxy anthraquinone (compound 8).
The present invention also provides an isolated and purified anthraquinone which has the formula: 
The present invention also provides an isolated and purified anthraquinone which has the formula: 
The present invention also provides an isolated and purified anthraquinone which has the formula: 
The present invention also provides an isolated and purified anthraquinone which has the formula: 
The present invention also provides an isolated and purified anthraquinone which has the formula: 
The present invention also provides an isolated and purified anthraquinone which has the formula: 
The present invention also provides an isolated and purified anthraquinone which has the formula: 
The present invention also provides an isolated and purified anthraquinone which has the formula: 
The present invention also provides an isolated and purified anthraquinone which has the formula: 
Objects
Therefore, it is an object of the present invention to provide compositions such as the anthraquinones disclosed herein which have antihelminthic activity.
It is further an object of the present invention to provide methods for using the anthraquinones to inhibit helminths infecting warm-blooded animals, including humans.
Further still, it is an object of the present invention to provide methods for using the anthraquinones to inhibit pathogenic trematodes such as those of the Schistosoma genus infecting warm-blooded animals, including humans.
These and other objects of the present invention will become increasingly apparent with reference to the following drawings and preferred embodiments.